Asbestos-induced toxicity remains to be a significant environmental condition. Despite strict regulatory controls to limit exposure, more than 1.3 million workers are exposed to hazardous levels of asbestos every year, which results in more than 100,000 deaths annually in the United States. One critical factor that contributes to the severity of toxicity in asbestos exposure is the generation of mitochondrial ROS (mtROS), which modulates alternative activation of lung macrophages; however, the molecular mechanism(s) regulating macrophage mtROS generation is not clearly defined. One of the NOX enzymes, NOX4, induces mtROS with various stimuli and in several cell types, but the modulation of the macrophage phenotype is not known to be mediated by NOX4. Our preliminary data show that lung macrophages from asbestos-injured subjects express high levels of the NOX4 gene compared to normal subjects. Inhibition or silencing NOX4 significantly abrogates mtROS. More importantly, the NOX1/4 inhibitor (GKT137831) abolishes alternative activation of macrophages. One important characteristic of alternatively activated macrophages is metabolic reprogramming from glycolytic metabolism to fatty acid oxidation, which is necessary to support long-term cellular activities. NOX1/4 inhibition attenuates asbestos-induced fatty acid oxidation. Similar observations were recapitulated in NOX4-/- mice. Lung macrophages from NOX4-/- mice displayed classical activation unlike the wild type mice, which had pro-fibrotic activation of macrophages. Furthermore, NOX4-/- mice were protected from asbestos- induced toxicity. Our hypothesis is that NOX4-mediated mtROS modulates metabolic reprogramming, alternative activation, and apoptosis resistance of lung macrophages, which promotes asbestos-induced toxicity. We will test this hypothesis with three specific aims. In Aim 1, the role of macrophage NOX4 in macrophage plasticity and in the pathogenesis of asbestos-induced toxicity will be tested in mice harboring a deletion of NOX4 in macrophages. Aim 2 will test the role of NOX4-derived mtROS in metabolic reprogramming and phenotypic plasticity using genetic approaches in asbestos-exposed macrophages. Aim 3 will test the role of NOX4 on the metabolism and phenotype of lung macrophages from asbestos-injured subjects ex vivo with GKT137831 and RNAi-mediated NOX4 silencing. These studies may uncover NOX4 as an ideal therapeutic target to attenuate asbestos-induced toxicity by modulating mitochondrial metabolism and macrophage plasticity.